For some years one of the prime objectives of gene technology has been the isolation and identification of genes. For this there is available a whole range of procedures, which have been directed in effect at the isolation, and identification of permanently expressed genes.
The isolation and/or identification of genes which are expressed only transiently, as for example the genes responsible for programmed cell death, cell-cycle genes, DNA repair genes and differentiation-specific genes, is much more difficult.
To identify such genes in mammalian cells, where genetic analysis by the use of Drosophila melanogaster is unsuitable, a process has been developed which is based on the induction of gene fusions between a reporter gene without promotor and the control elements of a cellular gene via specific vectors, which are described as "gene traps" or "promotor traps". Various types of vector have been developed for insertion mutagenesis in mammals, whereby a reporter gene is inserted into the chromosome at a large number of places depending on chance, including in transcriptionally active areas. During selection for gene expression, clones are retained in which the reporter gene is fused with the regulatory elements of an endogenous gene. In this way the vectors act as gene traps and provide a very helpful means for the analysis of gene function (Reviews in Hill & Wurst, 1993; Hill & Wurst 1993; von Melchner et al 1993; von Melchner & Ruley, 1995). In the majority of cases the gene-trapping vectors are transduced as recombinant retroviruses, although electroporated DNA is also used. The retroviruses display the advantage that they integrate in several areas throughout the entire genome and hence scarcely damage the neighbouring DNA (Varmus, 1988; Coffin et al., 1989; Goff, 1990; Sandmeyer et al., 1990; Withers/Ward et al., 1994).
It could be demonstrated that the gene traps are a practical means of analysing gene function in mice. Since totipotent mouse embryonic stem (ES) cells are used as cellular targets, mouse strains displaying inactivated gene function on account of mutations can be constructed. Unlike with gene splitting due to homologous recombination, the gene-trapping processes are not confined to genes, are sustainable for the cloned sequences and hence represent a process for the isolation and identification of genes as yet unknown.
Nevertheless, in order to identify and isolate genes which must first be induced in cells, that is, which are not continuously being transcribed, for example transient genes, such as genes responsible for programmed cell death, cell cycle genes, DNA repair genes and differentiation specific genes, an additional process is necessary, in order conclusively to identify the transient cellular promotor captured in the gene trap, for instance through a durable signal independent of the promotor activity. Approximately 50% of the genes which are switched off in ES cell lines following infection/electroporation by gene-trapping vectors, manifest recessive phenotypes in mice (Friedrich and Soriano, 1991; Skarnes et al., 1992; von Melchner et al., 1992). This frequency is ten times greater than that observed following accidental insertion of retroviruses or microinjection of DNA (Gridley et al., 1987; Jaenisch et al., 1985). Due to this high efficiency of gene inactivation it appears sensible to isolate cell lines which display integration in most of the genes expressed (2-4.times.10.sup.4). It is not very practical, however, to transfer all mutations into the original line; furthermore many mutations result in genes of more limited significance. Consequently it would be desirable to exchange mutagenized ES cell clones for mutations relevant to important biological processes or genes.
With reference to this, those strategies are especially suitable which involve the preliminary selection of ES cell clones from interesting in vitro mutations, and for this employ a reporter gene to identify mutations e.g. in differentiation-specific genes. Although cultivated ES cells might have expressed genes which are not expressed in vivo, in 12 cases fusion genes were found which were expressed in ES cells as well as in embryos (De Gregory et al., 1994; Reddy et al., 1992). It further appears to be the case that a change in the expression of fusion genes during differentiation in vitro very closely predicts the change in expression during in vivo development (Reddy et al., 1992). This is in general agreement with earlier observations, in which in vitro-regulated genes were also strictly regulated in vivo (Muthuchamy et al., 1993; Rappotee et al., 1988; Rogers et al., 1991; Scholer et al., 1990; Sharpe et al., 1990).